Circulating lipids in human plasma or lymphatic fluid consist of cholesterol, cholesteryl esters, triglycerides and phospholipids. These lipids are transported in large molecular complexes called lipoproteins, which consist of a core of cholesteryl esters and/or triglycerides, an envelope of phospholipids and free cholesterol, and apolipoproteins (Scriver et al., Eds., The Metabolic and Molecular Basis of Inherited Disease, 7th Ed., p. 1841-1851 (McGraw-Hill, New York 1995)). Apolipoproteins are involved in the assembly and secretion of the lipoprotein, as well as the activation of lipoprotein modifying enzymes, such as lecithin cholesterol acyl transferase (LCAT). In addition, apolipoproteins provide structural integrity and are ligands for a large spectrum of receptors and membrane docking proteins. The plasma lipoproteins are categorized into five types according to size: chylomicrons (largest in size and lowest in density), very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL), low density lipoproteins (LDL) and high density lipoprotein (HDL).
Chylomicrons, VLDLs, IDLs, and LDLs transport exogenous and endogenous cholesterol and triacylglycerols to peripheral sites, where the lipids play a role in various metabolic pathways and serve as a major constituent of cell membranes. Chylomicrons are assembled in the intestinal mucosa as a means to transport dietary cholesterol and triacylglycerols to various tissues. VLDLs are formed in the liver to transport endogenous cholesterol and triacylglycerols synthesized by the liver to extra-hepatic tissues, such as muscle and adipose tissue. In fasting serum, VLDLs contain 10-15% of the total serum cholesterol and most of the triglyceride. In circulation, VLDLs are converted to LDLs through the action of lipoprotein lipase. LDLs are the primary plasma carriers of cholesterol for delivery to all tissues, typically containing 60-70% of the total fasting serum cholesterol.
In contrast, HDLs are involved in “reverse cholesterol transport”, the pathway by which excess cholesterol is transported from peripheral sites back to the liver, where it is excreted in the form of bile salts (Glomset, J. A., J. Lipid Res., 9, 155-167 (1968)). Nascent HDLs are synthesized de novo in the liver and small intestine, as protein-rich disc-shaped particles devoid of cholesterol and cholesterol esters. In fact, a major function of HDLs is to act as circulating stores of apolipoproteins, primarily apo C-I, apo C-II, and apoE. The nascent or protein-rich HDLs are converted into spherical lipoprotein particles through the accumulation of cholesteryl esters obtained from cellular sources. The HDL normally contain 20-30% of the total fasting serum cholesterol.
According to current theories, the reverse efflux of cellular cholesterol to HDL is mediated through two mechanisms: an aqueous diffusion pathway and an apolipoprotein-mediated pathway. The relative importance of these distinguishable mechanisms depends on the cell type and metabolic state (Oram et al., J. Lipid Res., 37:2743-2491 (1996); Rothblat et al., J. Lipid Res., 40:781-796 (1999); Stein et al., Atherosclerosis, 144:285-301 (1999)). For many cells, the aqueous diffusion pathway is the principle pathway through which cholesterol efflux occurs (Johnson et al., Biochim. Biophys. Acta, 1085:273-298 (1991)). This pathway involves the bidirectional exchange of cholesterol between cell membranes and a lipoprotein acceptor, such as HDL, in the extracellular space through a process of passive transport (Remaley et al., Arterioscler. Thromb. Vasc. Biol., 17:1813-1821 (1997); Rothblat et al., J. Lip. Res., 40:781-796 (1999)). The exchange may occur primarily at surface microdomains known as caveolae (Fielding et al., Biochemistry, 34:14288-14292 91995)). Net efflux can be driven by conversion of cholesterol in the exteracellular compartment to cholesteryl ester by the action of LCAT.
Alternatively, in macrophage and fibroblast cells, cholesterol and phospholipid efflux is primarily mediated through apolipoproteins, such as apo A-I, apo A-II, and Apo E (Remaley, supra (1997); Francis, et al., J. Clin. Invest., 96:78-87 (1995); Vega et al., J. Intern. Med., 226:5-15 (1989); Sakar et al., Biochim. Biophys. Acta, 1438: 85-98 (1999); Hara et al., J. Biol. Chem., 266:3080-3086 (1991); Fielding et al., J. Lipid Res., 38, 1503-1521 (1997); Oram et al., J. Lipid Res., 37, 2743-2491 (1996)). The process of apolipoprotein-mediated lipid efflux particularly dominates in macrophages and other scavenger cells when they are cholesterol-loaded and/or growth-arrested. Apolipoprotein-mediated efflux is an active transport process that requires the direct interaction of the apolipoprotein with the cell surface, the lipidation of the apolipoprotein, and the subsequent dissociation of the lipid-apolipoprotein particle from the cell (Oram, supra (1996); Mendez, A. J., J. Lipid Res., 38, 1807-1821 (1997); Remaley, supra (1997); Mendez, A. J., J.Lipid Res., 37, 2510-2524 (1996)). Once removed from the cell, the cholesterol-rich HDL particles are transported to the liver and removed from the body as described.
Abnormal lipoprotein function and/or metabolism resulting from genetic defect or as a secondary effect of another disorder can have serious biological consequences. In addition to dietary influences, disorders such as diabetes, hypothyroidism, and liver disease can result in elevated plasma levels of LDL-cholesterol and triglycerides. Elevated levels of LDL-cholesterol and triglycerides have been identified as major risk factors associated with the incidence of coronary heart disease, which is the primary cause of death in the United States and other industrialized nations (Hokanson et al., J. Cardiovasc. Risk., 3:213-219 (1996); The Expert Panel, JAMA, 269:3015-3023 (1993)). The accumulation of excess LDL-cholesterol on arterial walls can lead to the formation of atherosclerotic plaques, which play a major role in the development of heart disease. A plaque is believed to form when free radicals released from arterial walls oxidize LDL. According to theory, the oxidized form of LDL triggers an inflammatory response, attracting circulating cells to the site which contribute to the formation of a lipid plaque. Among these are macrophages and other cells that contain scavenger receptors that accumulate cholesterol in an unregulated manner (Brown et al., Ann. Rev. Biochem., 52:223-261 (1986)). Vast stores of internal cholesterol result in conversion to a foam cell phenotype, which is believed to be a major contributor to the development of vascular lesions. As the plaque builds up, the arterial walls constrict, reducing blood flow to the heart.
Interestingly, however, an estimated 60% of heart attacks occur in persons who do not have elevated blood levels of LDL-cholesterol. Of these, an estimated 45% are associated with below average blood levels of HDL-cholesterol, indicating that low HDL-cholesterol level is a significant risk factor for coronary heart disease. In fact, recent studies have indicated that a decreased HDL-cholesterol level is the most common lipoprotein abnormality seen in patients with premature coronary artery disease (Genest J., Circulation, 85:2025-2033 (1992); Genest et al., Arterioscler. Thromb., 13:1728-1737 (1993)). Although the basis for the inverse association between HDL-cholesterol and coronary heart disease is not well understood, it has been suggested that the cardioprotective role of HDL may stem from its activity relating to the promotion of cholesterol efflux from macrophage foam cells in atherosclerotic lesions.
One example of cardiovascular disease associated with low HDL is Tangier disease (TD), a rare genetic disorder characterized by a near or complete absence of circulating HDL. In addition to near zero plasma levels of HDL, patients with TD have a massive deposition and accumulation of cholesteryl esters in several tissues, including tonsils, lymph nodes, liver, spleen, thymus, intestine, and Schwann cells (Fredrickson, D. S., J. Clin. Invest., 43, 228-236 (1964); Assmann et al., The Metabolic Basis of Inherited Disease, (McGraw-Hill, New York, 1995)). Although the cellular mechanisms have not been previously identified, recent studies have shown that cells from subjects with TD are defective in the process of apolipoprotein-mediated removal of cholesterol and phospholipids (Remaley et al., Arterioscler. Thromb. Vasc. Biol., 17, 1813-1821 (1997); Francis et al., J. Clin. Invest., 96, 78-87 (1995); Rogler et al., Arterioscler. Thromb. Vasc. Biol., 15, 683-690 (1995)). These results have led to the proposal that the severe HDL deficiency in TD patients stems from the inability of nascent apo A-I to acquire lipids. Because they do not mature into lipid-rich particles, the nascent HDL in TD patients is rapidly catabolized and removed from the plasma, resulting in the near zero levels of circulating HDL (Remaley, supra (1997); Francis, supra (1995); Horowitz et al., J. Clin. Invest., 91, 1743-1752 (1993); Schaefer et al., J. Lip. Res., 22:217-228 (1981)).
Other disorders associated with severe premature atherosclerosis and high risk for coronary heart disease resulting from diminished HDL-cholesterol levels are hypoalphalipoproteinemia and familial HDL deficiency syndrome (FHA). Persons with these disorders often have normal LDL-cholesterol and triglyceride levels. In addition, disorders such as diabetes, alcoholism, hypothyroidism, liver disease, and elevated blood pressure can result in diminished plasma levels of HDL-cholesterol, although many of these disorders are also accompanied by elevated LDL-cholesterol and triglceride levels.
Current treatments for coronary heart disease have focused primarily on diet manipulations and/or drug therapies aimed at lowering the plasma level of LDL-cholesterol by inhibiting LDL secretion or promoting LDL turnover. Derivatives of fibric acid, such as clofibrate, gemfibrozil, and fenofibrate, promote rapid VLDL turnover by activating lipoprotein lipase. Nicotinic acid reduces plasma levels of VLDL and LDL by inhibiting hepatic VLDL secretion. In addition, HMG-CoaA reductase inhibitors, such as mevinolin, mevastatin, pravastatin, simvastatin, fluvastatin, and lovastatin reduce plasma LDL levels by inhibiting the intracellular synthesis of cholesterol, which causes an increase in the cellular uptake of LDL. In addition, bile acid-binding resins, such as cholestyrine, colestipol and probucol decrease the level of LDL-cholesterol by increasing the catabolism of LDL-cholesterol in the liver.
However, many of these therapies are associated with low efficacy and/or side effects that may prevent long-term use. For example, use of HMG-CoaA reductase inhibitors carry a significant risk of toxicity because they inhibit the synthesis of mevalonate, which is required for the synthesis of other important isoprenoid compounds in addition to cholesterol. Also, gemfibrozil and nicotinic acid are associated with serious adverse effects, including renal injury, myopathy, myoglobinuria and intolerable skin flushing and itching. In addition, the role of probucol in treating patients with coronary heart disease is uncertain because its administration results in lower HDL-cholesterol levels as a side effect of reducing LDL-cholesterol.
Furthermore, treating patients who have isolated low HDL-cholesterol levels provides a particularly difficult therapeutic challenge. For instance, patients with Tangier disease exhibit a 4- to 6-fold increase in cardiovascular disease even though their LDL levels are already reduced by about 50%. While there is some evidence that gemfibrozil and nicotinic acid may simultaneously elevate HDL levels, in general, therapies aimed at lowering plasma LDL-cholesterol levels are not effective for Tangier patients who suffer from coronary heart disease as a result of diminished HDL levels. Likewise, patients with hypoalphalipoproteinemia, familial HDL deficiency syndrome, or other cardiovascular disease resulting from low levels of HDL will not benefit from therapies aimed at lowering the level of plasma LDL.
The problems associated with current therapies for cardiovascular disease stem partially from the fact that the biology involved in the movement of cholesterol in and out of cells is not fully understood. Furthermore, the proteins that play a role in cholesterol movement are not fully known. Therefore, there remains a need for a better understanding of cholesterol cell biology, as well as new methods for treating humans suffering from cardiovascular disease and other disorders associated with hypercholesterolemia. Additionally, there remains a need for new methods of diagnosing cardiovascular disease and new methods of screening patients to identify those at high risk for developing cardiovascular disease.
The identification of genes and proteins involved in cholesterol transport would be useful in the development of pharmaceutical agents for the treatment of heart disease and other disorders associated with hypercholesterolemia and atherosclerosis. In addition, the identification of such genes would be useful in the development of screening assays to screen for compounds that regulate the expression of genes associated with cholesterol transport. The identification of such regulatory compounds would be useful in the development of further therapeutic agents. Furthermore, the identification of genes and proteins involved in cholesterol transport would be useful as diagnostic indicators of cardiovascular disease and other disorders associated with hypercholesterolemia.